Identi ﬁ cation and characterization of auxin response factor (ARF) family members involved in ﬁ g ( Ficus carica L.) fruit development

The auxin response factor (ARF) combines with AuxREs cis-acting elements in response to auxin to regulate plant development. To date, no comprehensive analysis of ARF genes expressed during fruit development has been conducted for common ﬁ g ( Ficus carica L.). In this study, members of the FcARF gene family were screened, identi ﬁ ed in the ﬁ g genome database and their features characterized using bioinformatics. Twenty FcARF genes were clustered into three classes, with almost similar highly conserved DBD (B3-like DNA binding domain), AUX/IAA (auxin/ indole-3-acetic acid gene family) and MR domain structure among class members. Analysis of amino acid species in MR domain revealed 10 potential transcription activators and 10 transcription inhibitors, and 17 FcARF members were predicted to be located in the nucleus. DNA sequence analysis showed that the ARF gene family consisted of 4 – 25 exons, and the promoter region contained 16 cis-acting elements involved in stress response, hormone response and ﬂ avonoid biosynthesis. ARF genes were expressed in most tissues of ﬁ g, especially ﬂ ower and peel. Transcriptomics analysis results showed that FcARF2 , FcARF11 and FcARF12 , belonging to class-Ia, were stably and highly expressed in the early development stage of ﬂ ower and peel of ‘ Purple peel ’ ﬁ g. However, their expression levels decreased after maturity. Expression of class-Ic member FcARF3 conformed to the regularity of ﬁ g fruit development. These four potential transcription inhibitors may regulate fruit growth and development of ‘ Purple Peel ’ ﬁ g. This study provides comprehensive information on the ﬁ g ARF gene family, including gene structure, chromosome position, phylogenetic relationship and expression pattern. Our work provides a foundation for further research on auxin-mediated ﬁ g fruit development.

ARF proteins are negatively regulated by auxin/indole-3-acetic acid (Aux/IAA) proteins. The potential for interaction between ARF and Aux/IAA proteins is high as their C-terminal domains are homologous (Shen et al., 2010). Aux/IAA genes play a major role in regulating many auxin processes in plants. Aux/IAA genes exist as a family in many plants and are associated with leaf curl patterns, plant phototropism and tropism, plant height, and especially lateral root formation (Reed, 2001). In Arabidopsis thaliana, axr5/iaa1 mutant showed auxin resistance and loss of root and stem geotropism and hypocotyl phototropism (Yang et al., 2004). Mutations in AUXIN RESISTANT1 (AUX1)/ LIKE-AUX1 (LAX) genes result in auxin-related developmental defects and have been implicated in regulating root development, female gametophyte development and embryo development in Arabidopsis thaliana (Swarup & Bhosale, 2019). AtIAA8 regulates lateral root formation by interacting with auxin receptor AtTIR1 and ARF transcription factors in the nucleus (Arase et al., 2012). MdARF1 regulates anthocyanin accumulation in fruit by interacting with MdIAA121 in apples .
As an important plant growth regulator, the role of ARF gene family in fruit ripening and development has been investigated in many species but not in fig fruits. With its origins in the Mediterranean coastal region, the common fig (Ficus carica L.) is one of the widely cultivated ancient fruit trees in the world. The fig fruit (syconia), an aggregated fruit formed by the expansion of female flower tissue and receptacle, has high antioxidant activity, great taste and high nutritional value (Song et al., 2021). The fig fruit exhibits a typical double sigmoid growth curve, including two rapid growth phase (I and III) separated by slower growth phase (II) (Song et al., 2021). The fig has the advantages of short juvenile period, easy vegetative growth and reproduction, and is gradually becoming a new model plant for studying fruit development mechanisms . Presently, the genome-wide characterization of fig auxin signal transduction-related components and their expression patterns has not been carried out. In this study, genome-wide data of 'Horaishi' and 'Dottato' (Mori et al., 2017;Usai et al., 2020) and 'Purple Peel' fig transcriptome data at different developmental stages (NCBI accession number PRJNA723733) were used to predict ARF genes. In total, 20 ARF genes were identified in common fig, and their features, including chromosome position, phylogenetic relationship, gene structure and motif characteristics, characterized. In addition, we described the transcriptional accumulation patterns of ARF genes in different tissues, including at different developmental and maturation stages of female flowers and peel. Our study provides a good basis for further exploring the role of FcARFs and characterizing their functions.

Plant materials
The fig cultivar 'Purple Peel' plants were established at Shangzhuang Experimental Station of China Agricultural University, Beijing, China. The roots, stems, leaves of 'Purple Peel', and fruits at different developmental stages were collected for qRT-PCR analysis. Fruits were sampled at six stages based on the characteristics of fruit development. Stage 1 represented phase I (the first rapid growth period), stages 2, 3, and 4 were the early, middle, and late stages of phase II (slow growth period), and stages 5 and 6 represented phase III (the second rapid growth period). At each stage, 50 fruits were sampled. The peel and flower were separated onsite during sampling. Fresh samples were quick-frozen with liquid nitrogen and stored at −80 C until RNA extraction.

Multiple sequence alignment and phylogenetic analysis
Using Clustal 2.0, multiple-sequence alignments of amino acid sequences of predicted ARF genes in fig was carried out, with default parameters (Song et al., 2021). ClustalX 2.11 was used to analyze phylogenetic relationships among ARF gene families in Arabidopsis, fig, apple, grape and strawberry. (Liu et al., 2015;Luo et al., 2014;Sun, Fan & Ling, 2015). The phylogenetic tree of FcARFs was constructed using MEGA 7.0 via the Neighbor-Joining method with calibration test parameter "Bootstrap = 1,000 (1,000 replicates)" (Tamura et al., 2011;Kumar, Stecher & Tamura, 2016). The analysis involved 87 amino acid sequences and ARF gene families in five species including fig were divided following the division of ARF gene families in Arabidopsis thaliana. Percentage confidence values were shown on branches and the evolutionary distances used to infer the phylogenetic tree via Poisson correction method (Zuckerkandl & Pauling, 1965). Gaps were treated as a complete deletion. There were a total of 1,619 positions in the final dataset.

Gene structure and conserved motif analysis
FcARFs gene sequences were downloaded from fig genome database (https://www.ncbi. nlm.nih.gov/genome/?term=Ficus+carica), and the intron/exon structure map of the fig ARFs was generated online using the Gene Structure Display Server (GSDS: http://gsds.cbi. pku.edu.ch) (Guo et al., 2007). FcARFs amino acid sequences were referenced to fig genome database protein RefSeq. Because ARF family genes contain highly conserved domains, DNAMAN was used for multiple sequence alignment of FcARF protein sequences. The length and sequence information of other conserved motifs except the ARF conserved domain were obtained using the online MEME4.11. 2 tool (http://meme.sdsc. edu/meme/itro.html) with the following parameters: number of repetitions "any," highest motif number "15," motif length "6-100," and default values for the other parameters.

Chromosome mapping and collinearity analysis
Tandem replication of ARF genes usually refers to adjacent homologous genes on the same chromosome, whereas gene duplication events are regarded as repeated homologous genes on different chromosomes (Liu et al., 2011). Whether FcARF gene is considered an important event of tandem repeat between genes depends on its location on the chromosome. To study the distribution of ARF genes on chromosomes, their chromosomal location and annotation information were utilized. An openly available database and Map Draw software were used to generate physical location information for homolinear analysis of ARF genes in fig. The genome data of Ficus hispida and Ficus microcarpa were downloaded from the National Genomics Data Center database (https://ngdc.cncb.ac.cn/search/?dbId=bioproject&q=PRJCA002187&page=1) . An interspecies collinearity analysis of ARFs in fig, F. hispida, and F. macrocarpa was performed using MCscanX and TBtools (Song et al., 2021). The final map was generated with Circos version 0.63 (http://circos.ca/). The non-synonymous replacement rate (Ka) and synonymous replacement rate (Ks) of replicated gene pairs were calculated using KaKs_Calculator 2.0 , with Ka/Ks ratio taken as environmental selection pressure.

Protein functional connection network
Since fig species was not included in STRING database, the search species was set to Arabidopsis thaliana. The interaction networks of 20 ARF proteins were analyzed using the STRING protein interaction database (http://string-db.org/), which is an online protein-protein interaction networks functional enrichment analysis prediction site (Szklarczyk et al., 2021). Protein-protein interaction networks were built with an interaction score of −1 to 1. In total, 20 fig ARF proteins were aligned with AtARF proteins using Blastp alignment with E-value of 1e−0 (Rosas-Camacho, 2009;Chen et al., 2015). The interaction network of FcARF protein was constructed using homologous and Arabidopsis thaliana ARF protein, and genes of interest were visualized using Cytoscape software (Doncheva et al., 2019).

Expression analysis of FcARFs using RNA-seq and qRT-PCR
TBtools was used to analyze the expression patterns of FcARFs in 'Purple-Peel' fig fruit based on RNA-seq library (NCBI accession number PRJNA723733). Their peel and female flower tissue were collected at six stages of fruit development and significant differential expression was determined with p < 0.05 and |log2(fold change)| ≥ 1.
Different tissues and fruits were sampled from 'Purple Peel' plant at six stages for qRT-PCR. The peel and flower were separated during sampling. Fresh samples were quick-frozen with liquid nitrogen and stored at −80 C for RNA extraction. Total RNA was extracted using the CTAB method and reversed transcribed using reverse transcription kit (TaKaRa, Dalian, China). The specific primers for qRT-PCR are shown in Table S5. The design parameters for the primers include amplicon length, 100-150 bp; primer length, 15-25 bp; melting temperature (Tm), 57-61 C; GC base content, 40-60%; and excluding conserved domain (Li et al., 2019). Following a previously published method (Song et al., 2021), all qRT-PCR reaction volumes were set at 10 mL. The cycle parameters were as follows: pre-denaturation at 94 C for 15 s, annealing at 60 C for 30 s and extension at 72 C for 1 min, for 40 cycles. qRT-PCR reaction for each gene was replicated three times. Fc18S and FcActin were used as an internal standard to calculate relative fold differences based on comparative cycle threshold values. We calculated relative expression levels using the comparative Ct (2 −ΔΔCt ) method. SPSS 26.0 software was used for significance analysis.

ARF family members identified in fig
Twenty ARF genes were identified in published fig genome and named FcARF1 to FcARF20 following the equivalent classification for Arabidopsis thaliana ( Fig. 1A and Table 1). Detailed information for each candidate gene, including gene name, gene ID, number of exons, protein length, protein molecular weight (kDa), isoelectric point (pI), instability coefficient, Aliphatic index, GRAVY and location in cells, is shown in Table 1. The length of amino acids encoded by FcARFs ranged from 588 (FcARF10) to 1,150 (FcARF7), averaging 782.95 amino acids. The number of exons and predicted molecular weight ranged from three (FcARF10) to 17 (FcARF18) and 52.73 (FcARF4) to 127.06 KDa (FcARF7), respectively. The isoelectric point (pI) ranged from 5.03 (FcARF5) to 9.52 (FcARF11), and acidic amino acids were enriched in the protein molecules. In total, 80.95% of fig ARF family proteins had isoelectric points less than seven, with most of them being weakly acidic, consistent with pl of ARF proteins in citrus and strawberry Wang et al., 2019). On average, the total hydrophilicity of ARF family proteins in fig was negative, ranging from −0.726 (FcARF11) to −0.322 (FcARF17), which indicated that these proteins were hydrophilic. The instability index ranged from 42.05 (FcARF17) to 72.35 (FcARF15), suggesting that all these proteins were stable (II > 40). The Aliphatic index ranged from 63.06 (FcARF11) to 84.46 (FcARF18). Twenty FcARF proteins were localized in subcellular structures by online software Target P and CELLO v2.5. The results showed that they were mostly distributed in the nucleus and cytoplasm.

Phylogenetic analysis of FcARFs
In total, 87 ARF proteins comprising 23 from Arabidopsis thaliana, 20 from fig, 12 from strawberry, 19 from grape and 13 from apple were obtained from NCBI database to construct a phylogenetic tree using Neighbor-Joining approach. FcARF proteins were divided into three classes (Fig. 1). Class-I was further divided into three classes, including class-Ia with the following five members: FcARF1, FcARF2, FcARF9, FcARF11 and FcARF18. Class-Ib included three members (FcARF12, FcARF14 and FcARF20) whereas class-Ic included two (FcARF3 and FcARF4). Class-II had six members and class-III had four. Compared with the equivalent Arabidopsis class, the number of ARF members in fig class-Ib was less. These results indicated that gene loss or gain occurred during the evolution of ARF family genes in fig.

Analysis of ARF gene family structure
Typical ARF protein has three conserved domains: N-terminal B3-like DNA binding domain (DBD), C-terminal dimerization domain (CTD), and a middle region (MR) (Xing et al., 2011). Figure 2 shows that all FcARFs contained MR as an intermediate domain. All proteins contained a complete CTD structure except class-III members. Similarly, DBD structure existed in all FcARFs proteins except FcARF18. The branches of the same phylogenetic tree always had similar structures, but some genes in the same class exhibited different gene structures. Among them, four FcARFs (FcARF17, FcARF10, FcARF13 and FcARF16) had truncated CTD domains distributed in class-III of the phylogenetic tree ( Fig. 2A). To exhaustively verify the accuracy of FcARF conserved domains, the amino acid sequence alignment results of FcARFs and identified the location of conserved domain were aligned using DNAMAN software (Fig. S1). The active MR domain has been found to be rich in glutamine (Q) whereas the inhibitory domain is rich in proline (P), serine (S) and threonine (T) (Wu et al., 2011;Tiwari, Hagen & Guilfoyle, 2003). Considering these characteristics, we analyzed the amino acid sequence and composition of the MR domain (Fig. S2). Subsequently, we predicted 10 potential transcription activators (FcARF4, FcARF5, FcARF7, FcARF9, FcARF10, FcARF13, FcARF16, FcARF17, FcARF18 and FcARF19) and 10 inhibitors (FcARF1, FcARF2, FcARF3, FcARF6, FcARF8, FcARF11, FcARF12, FcARF14, FcARF15 and FcARF20) (Fig. 2B).

Conservative FcARFs motif and cis-acting element analysis
The exon-intron structures of genes in the same gene family are similar, reflecting the close evolutionary relationship among all its members. The number of introns in FcARF ranged from three to 17 (Fig. 3A). FcARF18 gene in class-Ia had the most introns (17), whereas class-III members contained the least, suggesting that members have experienced intron loss during evolution. Conserved motifs 1-15 of FcARFs were analyzed (Table S3, Fig. S3). Motif 2 and motif 8 belonging to DBD and MR domains, respectively, were highly conserved in all FcARF proteins. Our analysis of conserved motifs in the database revealed that conserved motifs in FcARF are unstable, and their positions had changed except motif 2 and motif 8. In addition, motifs 15 only existed in specific genes, which agrees with previously reported results (Wei et al., 2016). This indicates that these motifs may be essential for specific regulatory functions of these genes.
We found 2 kb sequence upstream of the initiation codon of FcARF gene in PLACE database that predicted many cis-acting elements larger than 4 bp. TGA-element and TGA-box were associated with auxin response and transcriptional activation. There were at least 16 cis-acting regulatory elements in the promoter region of FcARF family members in fig (Fig. 3B). They were involved in abiotic stress responses, including light response, anaerobic inducibility, anoxic specific inducibility, defense and stress response, drought induction, low temperature responsive and wound response. In addition, these cis-acting regulatory elements were involved in hormone response, (abscisic acid response, auxin response, gibberellin response, MeJA response and salicylic acid response), circadian control, flavonoid biosynthesis regulation, meristem expression and other regulatory elements. Drought stress-related MYBHv1 binding sites were found in the promoter regions of three ARF genes (FcbARF2, FcARF14 and FcARF18).  Fig. S4. FcARF1-FcARF20 were unevenly distributed in 12 chromosomes, apart from chromosomes 2 and 4. Chromosomes 1, 6, 7, 10, 11 and 12 had one ARF gene each, which were FcARF6, FcARF7, FcARF5, FcARF9, FcARF14 and FcARF20, respectively. Chromosomes 3, 5 and 9 had 3 ARF genes each, which were the most abundant. Tandem replication occurs when the distance between genes is less than 100 kb and exists on the same chromosomes, only FcARF3 and FcARF16 on Chr3 occurred. The FcARF gene family had three pairs of genes from fragment replication (Fig. 4A)

Protein interaction network of FcARFs
The ARF protein interacts with other proteins, such as IAA and AUX1 proteins, to regulate downstream gene expressions and exert biological functions. Correlation analysis of ARF proteins and interacting proteins in figs, as well as the gene expression correlation network (Song et al., 2022), was analyzed based on gene expressions in the developmental stages of fig fruits (Fig. 5B). A protein interaction network was constructed by STRING, which included 5 ARF proteins and nine other proteins (5 IAA proteins). Expressions of the five FcARFs were positively correlated with each other, co-expression scores were all above 0.7, with FcIAA1, FcBZR1 and FcAUX1 being negatively correlated with expressions of the five FcARFs.
In Fig. 5A, thickness of lines represents protein interaction strengths. IAA8 interacted with five FcARFs, and their expressions were positively correlated, including FcARF15-IAA8. In Arabidopsis, interactions of AtARF15 and AtIAA8 proteins respond to early auxin responses and cooperatively regulates lateral root growth, development as well as morphogenesis (Arase et al., 2012). There was a strong interaction between ARF6-PIF4, with an expression correlation coefficient of 0.808, implying a positive correlation. Direct inhibition of auxin-responsive gene expressions mediated by interactions of AtPIF4 and AtARF6 in Arabidopsis constitutes a new layer in regulatory mechanisms of photoinhibition of auxin-induced hypocotyl elongation (Oh et al., 2014). IAA1 was predicted to interact with FcARF8 and FcARF19, and expression correlation indices were −0.582 and −0.571, respectively, implying negative correlations. This is consistent with the The expression levels and interaction coefficients predict that FcIAA8 is a key positive cooperative factor in the network, while IAA1 is a negative regulatory cooperative factor.

Expressions of FcARFs during fig fruit ripening
Expressions patterns of FcARFs in 'Purple peel' fig peel and flower tissues at different growth stages were constructed by TBtools (Fig. 7). Based on FPKM values, they were divided into four groups, among which four members greater than 100 came from three classes, respectively. Class-I had four members (FcARF2, FcARF3, FcARF11, and FcARF12) expressed in early stages of flower and peel development. The 50-100 group consists of three members (FcARF1, FcARF8 and FcARF18) from class-Ia and class-II. Expression patterns of FcARF1 and FcARF18 were consistent, being highest in early stages of female flower development. The rest of the members with FPKM values between 10 and 50. During the six growth stages of female flowers and peel, FcARF3 from class-Ic was stably expressed, with FPKM values >50. In addition, expressions of FcARF3 in fast-growing phase I and III were higher than those in slow-growing phase II. Expressions of class-Ia members FcARF2/9/11 were highest in the early developmental stages of fruits and were decreased after maturity. FcARF11 levels were highest at P1, P2 and P4, while   19,20) have DBD and MR domains that determine their functions based on amino acid compositions Wang et al., 2007;Okushima et al., 2005). Through domain comparisons ( Fig. S3 and Table S3), it can be found that FcARF18 lacks motif 2 on the DBD domain, which inhibits its ability to recognize and bind DNA by forming a helix-turn-helix domain (Tiwari, Hagen & Guilfoyle, 2003;Roosjen, Paque & Weijers, 2018). In addition, most plant ARF gene families have an ARF protein without the CTD domain. All class-III FcARFs have truncated CTD domains, which is similar in Arabidopsis thaliana and other species ( Fig. 2A). Responses and signal transduction of plant cells to auxin are mainly mediated by the TIR1/AFB/-Aux/IAA-ARF signaling pathway in the nucleus. ARF participates in the auxin signaling pathway by forming a heterodimer with CTD regions of the Aux/IAA protein. The C-terminal domains of proteins are homologous. Without a complete CTD domain, FcARF10/13/16/17 may not participate in the auxin signaling pathway or perform its independent functions (Liu et al., 2011).
The ARF genes are transcriptional activators or inhibitors. The activation domain (AD) in glutamine (Q) and leucine (L) in the middle region of the ARF protein can act as a transcription activator, while the repression domain (RD) rich in proline (P), serine (S) and threonine (T) can act as a transcription inhibitor (Tiwari, Hagen & Guilfoyle, 2003;Zouine et al., 2014). In this study, the intermediate region of transcription factors in class-Ia and class-II was found to be rich in glutamine (Q), and it is postulated that class-II members have transcriptional activation functions. AtARF5, AtARF6, AtARF7, AtARF8 and AtARF19 in Arabidopsis thaliana, which are homologous to fig class-II, are ARF ClassA transcription activators. Middle regions of classes-I and -III are rich in proline (P), serine (S) and threonine (T). Therefore, members of class-Ia and class-II inhibit transcription. Our findings should be verified by studies of regulatory mechanisms (Roosjen, Paque & Weijers, 2018).

Evolution and gene structures of the ARF family
Gene replication, including tandem repeat and large fragment replication, is the most important mechanism of gene recombination and amplification (Vision, Brown & Tanksley, 2000). Tandem repeat refers to two adjacent genes on the same chromosome, while large fragment replication events occur on different chromosomes, which is an important way of gene expansion and species evolution (Liu et al., 2011;Xing et al., 2011). Collateral gene pairs (FcARF13 and FcARF10) were located on chromosome 1, while FcARF19, FcARF20, FcARF13 and FcARF17 were located on chromosomes 9, 11, 13 and 3, respectively. The former belongs to tandem replication, while the latter belongs to large fragment replication. In this study, 3 (15%) of 20 FcARFs were established to be involved in tandem repeat or large fragment replication events, while 33% of MdARFs in apples were associated with gene replication (Luo et al., 2014). Even though there are few gene replication events in fig, we postulated that gene replication is the main mode of ARF gene family amplification, implying that different types of ARFs gene family members have different evolutionary paths and rates.
FcARFs are mainly involved in plant hormone responses, growth regulation, light response and environmental stress responses. As trans-acting factors, hormones can bind hormone response elements in promoter sequences, thereby regulating the transcriptional activities of target genes, and can participate in plant growth as well as environmental stress processes, such as salt and temperature as signal transduction factors (Gunes et al., 2007). We found that the ARF gene family has various hormone-responsive elements, hat hormones induce ARF gene family expressions in fig, participating in plant growth and development.
Fruit development and ripening is a coordinated process of reproductive organ cell division, differentiation and expansion that is regulated by transcriptional regulatory networks (Uchiumi & Okamoto, 2010). Auxin promotes the growth of female flowers as well as receptacles and causes fruit enlargement that plays a key role in fruit ripening (Flaishman, Rodov & Stover, 2008;Kumar, Tyagi & Sharma, 2011). In this study, all FcARFs were expressed from the first stage (phase I) to sixth stage (end of phase III) of fruit development. The fruits of figs went through a fast-slow-fast developmental period. Sensing and transmitting signals have been shown to reveal changes in auxin expression patterns (Estrada-Johnson et al., 2017). AtARF2 has an important role in regulating flower formation as well as senescence of Arabidopsis thaliana (Ellis et al., 2005). Its homologous gene (FcARF2) was highly expressed in the early flower and peel development stages, which may regulate fig flower morphologies. FcARF11 and FcARF12 from class-Ia were abundantly expressed during the early stages of fig fruit female flower as well as peel development and were predicted to function as transcriptional repressors (Fig. 2B). It has not been established whether FcARF inhibition acts synergistically with other TFs.
AtARF3 has a major role in nutrition and reproductive development, regulation of gene and embryonic development, and determining the morphological patterns of pistil (Harper et al., 2000;Sessions et al., 1997;Xiao et al., 2004). Its homologous gene (FcARF3

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This study was supported by the National Natural Science Foundation of China (31372007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors: National Natural Science Foundation of China: 31372007.